Mandy L. Y. Sin

Antimicrobial susceptibility testing using high surface-to-volume ratio microchannels.

This study reports the use of microfluidics, which intrinsically has a large surface-to-volume ratio, toward rapid antimicrobial susceptibility testing at the point of care. By observing the growth of uropathogenic Escherichia coli in gas permeable polymeric microchannels with different dimensions, we demonstrate that the large surface-to-volume ratio of microfluidic systems facilitates rapid growth of bacteria. For microchannels with 250 microm or less in depth, the effective oxygenation can sustain the growth of E. coli to over 10(9) cfu/mL without external agitation or oxygenation, which eliminates the requirement of bulky instrumentation and facilitates rapid bacterial growth for antimicrobial susceptibility testing at the point of care. The applicability of microfluidic rapid antimicrobial susceptibility testing is demonstrated in culture media and in urine with clinical bacterial isolates that have different antimicrobial resistance profiles. The antimicrobial resistance pattern can be determined as rapidly as 2 h compared to days in standard clinical procedures facilitating diagnostics at the point of care.

Hybrid electrokinetic manipulation in high-conductivity media

This study reports a hybrid electrokinetic technique for label-free manipulation of pathogenic bacteria in biological samples toward medical diagnostic applications. While most electrokinetic techniques only function in low-conductivity buffers, hybrid electrokinetics enables effective operation in high-conductivity samples, such as physiological fluids (∼1 S m(-1)). The hybrid electrokinetic technique combines short-range electrophoresis and dielectrophoresis, and long-range AC electrothermal flow to improve its effectiveness. The major technical hurdle of electrode instability for manipulating high conductivity samples is tackled by using a Ti-Au-Ti sandwich electrode and a 3-parallel-electrode configuration is designed for continuous isolation of bacteria. The device operates directly with biological samples including urine and buffy coats. We show that pathogenic bacteria and biowarfare agents can be concentrated for over 3 orders of magnitude using hybrid electrokinetics.

Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis

Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but also more informative than the current standard.

System Integration - A Major Step toward Lab on a Chip

Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.

Electrothermal Fluid Manipulation of High-Conductivity Samples for Laboratory Automation Applications.

Electrothermal flow is a promising technique in microfluidic manipulation toward laboratory automation applications, such as clinical diagnostics and high-throughput drug screening. Despite the potential of electrothermal flow in biomedical applications, relatively little is known about electrothermal manipulation of highly conductive samples, such as physiological fluids and buffer solutions. In this study, the characteristics and challenges of electrothermal manipulation of fluid samples with different conductivities were investigated systematically. Electrothermal flow was shown to create fluid motion for samples with a wide range of conductivity when the driving frequency was greater than 100 kHz. For samples with low conductivities (below I S/m), the characteristics of the electrothermal fluid motions were in quantitative agreement with the theory. For samples with high conductivities (greater than l S/m), the fluid motion appeared to deviate from the model as a result of potential electrochemical reactions and other electrothermal effects. These effects should be taken into consideration for electrothermal manipulation of biological samples with high conductivities. This study will provide insights in designing microfluidic devices for electrokinetic manipulation of biological samples toward laboratory automation applications in the future.

In Situ Electrokinetic Enhancement for Self-Assembled-Monolayer- Based Electrochemical Biosensing

This study reports a multifunctional electrode approach which directly implements electrokinetic enhancement on a self-assembled-monolayer-based electrochemical sensor for point-of-care diagnostics. Using urinary tract infections as a model system, we demonstrate that electrokinetic enhancement, which involves in situ stirring and heating, can enhance the sensitivity of the strain specific 16S rRNA hybridization assay for 1 order of magnitude and accelerate the time-limiting incubation step with a 6-fold reduction in the incubation time. Since the same electrode platform is used for both electrochemical signal enhancement and electrochemical sensing, the multifunctional electrode approach provides a highly effective strategy toward fully integrated lab-on-a-chip systems for various biomedical applications.

Hybrid electrokinetics for separation, mixing, and concentration of colloidal particles

The advent of nanotechnology has facilitated the preparation of colloidal particles with adjustable sizes and the control of their size-dependent properties. Physical manipulation, such as separation, mixing, and concentration, of these colloidal particles represents an essential step for fully utilizing their potential in a wide spectrum of nanotechnology applications. In this study, we investigate hybrid electrokinetics, the combination of dielectrophoresis and electrohydrodynamics, for active manipulation of colloidal particles ranging from nanometers to micrometers in size. A concentric electrode configuration, which is optimized for generating electrohydrodynamic flow, has been designed to elucidate the effectiveness of hybrid electrokinetics and define the operating regimes for different microfluidic operations. The results indicate that the relative importance of electrohydrodynamics increases with decreasing particle size as predicted by a scaling analysis and that electrohydrodynamics is pivotal for manipulating nanoscale particles. Using the concentric electrodes, we demonstrate separation, mixing, and concentration of colloidal particles by adjusting the relative strengths of different electrokinetic phenomena. The effectiveness of hybrid electrokinetics indicates its potential to serve as a generic technique for active manipulation of colloidal particles in various nanotechnology applications.

A Universal Electrode Approach for Automated Electrochemical Molecular Analyses

Transforming microfluidics-based biosensing systems from laboratory research into clinical reality remains an elusive goal despite decades of intensive research. A fundamental obstacle for the development of fully automated microfluidic diagnostic systems is the lack of an effective strategy for combining pumping, sample preparation, and detection modules into an integrated biosensing platform. Herein, we report a universal electrode approach, which incorporates dc electrolytic pumping, ac electrokinetic sample preparation, and self-assembled monolayer based electrochemical sensing on a single microfluidic platform, to automate complicated molecular analysis procedures that will enable biosensing applications in nontraditional health care settings. Using the universal electrode approach, major microfluidic operations required in molecular analyses, such as pumping, mixing, washing, and sensing, can be performed in a single platform. We demonstrate the universal electrode platform for detecting bacterial 16S rRNA, a phylogenetic marker, toward rapid diagnostics of urinary tract infection. Since only electronic interfaces are required to operate the platform, the universal electrode approach represents an effective system integration strategy to realize the potential of microfluidics in molecular diagnostics at the point of care.

Integrated Biosensor Assay for Rapid Uropathogen Identification and Phenotypic Antimicrobial Susceptibility Testing

BACKGROUND Standard diagnosis of urinary tract infection (UTI) via urine culture for pathogen identification (ID) and antimicrobial susceptibility testing (AST) takes 2-3 d. This delay results in empiric treatment and contributes to the misuse of antibiotics and the rise of resistant pathogens. A rapid diagnostic test for UTI may improve patient care and antibiotic stewardship. OBJECTIVE To develop and validate an integrated biosensor assay for UTI diagnosis, including pathogen ID and AST, with determination of the minimum inhibitory concentration (MIC) for ciprofloxacin. DESIGN, SETTING, AND PARTICIPANTS Urine samples positive for Enterobacteriaceae (n=84) or culture-negative (n=23) were obtained from the Stanford Clinical Microbiology Laboratory between November 2013 and September 2014. Each sample was diluted and cultured for 5h with and without ciprofloxacin, followed by quantitative detection of bacterial 16S rRNA using a single electrochemical biosensor array functionalized with a panel of complementary DNA probes. Pathogen ID was determined using universal bacterial, Enterobacteriaceae (EB), and pathogen-specific probes. Phenotypic AST with ciprofloxacin MIC was determined using an EB probe to measure 16S rRNA levels as a function of bacterial growth. MEASUREMENTS Electrochemical signals for pathogen ID at 6 SD over background were considered positive. An MIC signal of 0.4 log units lower than the no-antibiotic control indicated sensitivity. Results were compared to clinical microbiology reports. RESULTS AND LIMITATIONS For pathogen ID, the assay had 98.5% sensitivity, 96.6% specificity, 93.0% positive predictive value, and 99.3% negative predictive value. For ciprofloxacin MIC the categorical and essential agreement was 97.6%. Further automation, testing of additional pathogens and antibiotics, and a full prospective study will be necessary for translation to clinical use. CONCLUSIONS The integrated biosensor platform achieved microbiological results including MIC comparable to standard culture in a significantly shorter assay time. Further assay automation will allow clinical translation for rapid molecular diagnosis of UTI. PATIENT SUMMARY We have developed and validated a biosensor test for rapid diagnosis of urinary tract infections. Clinical translation of this device has the potential to significantly expedite and improve treatment of urinary tract infections.

Scaling analysis of a universal electrode for molecular biosensors

Nanoscale and molecular manipulation techniques, such as concentration, are crucial for the success of an automated point-of-care diagnostic system. Here, we report the development of an electrokinetic concentrator for integrated bioanalytical systems. By combining different electrokinetic forces, the concentrator is capable of concentrating nanoscale particles in less than 1 min and increasing the concentration by 2 orders of magnitude. The concentrator used a universal electrode design developed for a versatile electrochemical sensor platform. We performed a scaling analysis to generalize and optimize the design of the universal electrode. The data collapse analysis showed that the concentration process follows a universal kinetics, and the time scaling factor (T) scales with the effective electric field (E) as T ~ E0.48plusmn0.15 while the intensity scaling factor (T) increases with the effective electric field (E) as I ~ E0.93plusmn0.12.